The present disclosure provides cysteine-engineered antibodies and Fc fusion protein, and conjugate compounds comprising such cysteine-engineered molecules. Such conjugates can be utilized for diagnostic and therapeutic applications.
The use of antibodies has been established for the diagnosis and targeted treatment of patients with cancer, immunological and angiogenic disorders. The use of antibody-drug conjugates (ADC), i.e., immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit tumor cells in the treatment of cancer (Lambert (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) Nature Biotechnology 23:1137-1146; Payne (2003) Cancer Cell 3:207-21) theoretically allows targeted delivery of the drug moiety to tumors, where they bind to the target and may be internalized resulting in intracellular accumulation therein, where systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated. Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug-linking and drug-releasing properties (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). These methods include the conjugation of antibodies to drugs, toxins, radioisotopes, peptides, other antibodies, etc.
Conventional means of attaching, i.e., linking through covalent bonds, a drug moiety to an antibody generally leads to a heterogeneous mixture of molecules where the drug moieties are attached at a number of sites on the antibody. For example, cytotoxic drugs have typically been conjugated to antibodies through the often-numerous lysine residues of an antibody, generating a heterogeneous antibody-drug conjugate mixture.
Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent attachments to ligands or to form new intramolecular disulfide bonds. However, engineering cysteine thiol groups by the mutation of various amino acid residues of a protein to cysteine amino acids is potentially problematic, particularly in the case of unpaired (free cysteines) residues or those that are relatively accessible for reaction or oxidation. For example, formation of intramolecular or intermolecular disulfides can cause protein aggregation. The location of the engineered cysteine can affect the accessibility of the drugs during conjugation resulting in low yields. The introduction of new cysteines can render the antibody inactive or cause loss of binding specificity to its target due to misfolding or loss of tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9). Also, the conjugated compounds can have poor serum stability, leading to loss of activity and degradation (e.g., by proteolytic degradation or clearance of the antibody moiety, or by hydrolysis of the drug moiety). Thus, it is an object of the present disclosure to provide improved cysteine engineering strategies capable of yielding conjugate compounds with enhanced stability, e.g., serum stability.